The current blood bank system has inherent risks and serious limitations. Blood typing errors, immunogenicity, transmission of bacterial agents, and vital infections such as HIV-1 and hepatitis, pose life threatening dangers to transfusion patients. In addition, the limited availability of donors, the requirement for specific blood types, the short shelf-life of red blood cells, and the need for refrigeration all limit the accessibility of transfusions to patients. Development of a stable blood substitute could eliminate the risks of the current blood bank system and increase the availability of transfusions to patients in most environments. Thus, the delivery of oxygen (O2) to organs and tissues to alleviate symptoms due to blood loss or hypoxia is a major therapeutic goal.
No hemoglobin-based therapies have been approved for use in humans in the U.S. Potential therapies include a variety of artificial O2 carriers (reviewed by Spahn, D. R. et al. (20.05). “Artificial O2 carriers: status in 2005,” Curr. Pharm. Des. 11(31):4099-4114), such as engineered hemoglobins (e.g., U.S. Pat. No. 6,022,849). However, some potential blood substitutes, such as hemoglobin-based blood substitutes, are limited due to their reactivity with nitric oxide (NO). In particular, NO acts as a chemical messenger in the control of many important processes in vivo, including neurotransmission, inflammation, platelet aggregation, and regulation of gastrointestinal and vascular smooth muscle tone. NO reacts directly with O2 that is bound to hemoglobin to form methemoglobin and nitrate. Both the heme iron and NO become oxidized by the bound oxygen atoms, and the reaction occurs so rapidly that no replacement of O2 by NO is observed (see, e.g., U.S. Pat. No. 6,455,676).
Since NO is produced and consumed on a continuous basis, there is a natural turnover of NO in vivo. When cell-free hemoglobin is administered, the balance between NO production and consumption is altered by reactions with cell-free hemoglobin. The oxidative reaction between NO and O2 bound to hemoglobin is irreversible, resulting in the destruction of NO, O2, and hemoglobin. NO binding to hemoglobin without O2 bound is effectively irreversible on physiologic timescales since the half-life for dissociation of nitrosylhemoglobin is 5-6 hours, thereby effectively inactivating hemoglobin as a cell-free O2 carrier.
Once an NO molecule reacts with hemoglobin, it is eliminated from the pool of signal molecules, thereby causing certain adverse conditions. For example, the binding of NO to hemoglobin (with or without O2 bound) can prevent vascular relaxation and potentially lead to hypertension, which is sometimes observed after the administration of certain extracellular hemoglobin solutions.
NO is also needed to mediate certain inflammatory responses. For example, NO produced by the endothelium inhibits platelet aggregation. Consequently, as NO is bound by cell-free hemoglobin (with or without O2 bound), platelet aggregation may increase. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A2 and serotonin. These compounds may act synergistically with the reduced NO levels caused by hemoglobin scavenging to produce significant vasoconstriction. In addition to inhibiting platelet aggregation, NO also inhibits neutrophil attachment to cell walls, which in turn can lead to cell wall damage. Endothelial cell wall damage has been observed with the infusion of certain hemoglobin solutions.
Another major drawback of hemoglobin-based blood substitutes is their high affinity for O2. This high affinity limits the ability of hemoglobin to release oxygen at a clinically useful rate in desired locations (such as peripheral tissues). Alternatively, the release of O2 by lower affinity hemoglobin-based blood substitutes in arteries before reaching microvascular beds may cause vasoconstriction due to a hyperoxic vasoconstrictor response (Winslow hypothesis). Additionally, hemoglobin-based blood substitutes are hindered by the rapid clearance of cell-free hemoglobin from plasma due the presence of receptors for hemoglobin that remove cell-free hemoglobin from plasma. Cell-free hemoglobin may also cause kidney toxicity, possibly due to NO depletion in glomeruli, causing constriction and subsequent dysfunction.
Due to the limitations of current blood substitutes and the chronic shortage of donated blood, there remains a significant interest in and need for additional or alternative therapies for delivering oxygen. In particular, blood substitutes with a lower NO reactivity and/or a longer plasma retention time are desired. Oxygen carriers with dissociation constants or off rates for O2 binding that are appropriate for particular clinical or industrial applications are also needed. An exemplary industry application for which O2 carriers are desirable includes the growth of cells in culture, which is often limited by the amount of O2 that reaches the cells.